No Evidence for Clonal Selection Due to Lentiviral Integration Sites in Human Induced Pluripotent Stem Cells

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Stem Cells (Impact Factor: 6.52). 04/2010; 28(4):687-94. DOI: 10.1002/stem.322
Source: PubMed


Derivation of induced pluripotent stem (iPS) cells requires the expression of defined transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28) in the targeted cells. Lentiviral or standard retroviral gene transfer remains the most robust and commonly used approach. Low reprogramming frequency overall, and the higher efficiency of derivation utilizing integrating vectors compared to more recent nonviral approaches, suggests that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. We provide for the first time an extensive analysis of the lentiviral integration profile in human iPS cells. We identified a total of 78 independent IS in eight recently established iPS cell lines derived from either human fetal fibroblasts or newborn foreskin fibroblasts after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28. The number of IS ranged from 5 to 15 IS per individual iPS clone, and 75 IS could be assigned to a unique chromosomal location. The different iPS clones had no IS in common. Expression analysis as well as extensive bioinformatic analysis did not reveal functional concordance of the lentiviral targeted genes between the different clones. Interestingly, in six of the eight iPS clones, some of the IS were found in pairs, integrated into the same chromosomal location within six base pairs of each other or in very close proximity. Our study supports recent reports that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes.

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Available from: Thomas Winkler, Jul 30, 2014
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    • "One potential source of this clonal variation if integrative reprogramming methods have been used might be insertional mutagenesis caused by the reprogramming factors [10] [11]. However, the genomic integrity of iPSCs is frequently not assessed, despite studies demonstrating that the integration of viral vectors can result in the dysregulation of adjacent genes [12] [13]. This altered gene expression could lead to perturbed signalling pathways that may alter the pluripotency or differentiation potential of the iPSCs, similar to that observed in some virally-transduced hematopoietic stem cell lines [14]. "
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    • "The cloning of Dolly the sheep in 1997 dramatically changed the view of scientists on pluripotency and differentiation. The birth of Dolly clearly demonstrated that the nucleus of a somatic donor cell can be reprogrammed to a pluripotent state by factors present in the cytoplasm of an enucleated oocyte and the reconstructed embryo can ultimately lead to the development of a new organism (Wilmut et al. 1997). Since then, cloning has resulted in the successful production of live cloned offspring in more than 16 species, including cattle (Kato et al. 2000), mouse (Wakayama et al. 1998), goat (Baguisi et al. 1999), pig (Onishi et al. 2000; Polejaeva et al. 2000), cat (Shin et al. 2002), rabbit (Chesné et al. 2002), mule (Woods et al. 2003), horse (Galli et al. 2003), rat (Zhou et al. 2003), dog (Lee et al. 2005), ferret (Li et al. 2006a), red deer (Berg et al. 2007), buffalo (Shi et al. 2007), gray wolf (Oh et al. 2008) and camel (Wani et al. 2010). "
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    • "Notably, experiments with cells from " reprogrammable " mice support the conclusion that insertional mutagenesis is not a key driver of reprogramming as predicted from nonintegrative reprogramming studies and mapping of viral insertion sites (Aoi et al., 2008; Varas et al., 2009; Winkler et al., 2010). Consequently, one of the key questions in the reprogramming field has been whether only a particular subset of cells within the starting population, for example only those cells with a more undifferentiated state such as adult stem cells or progenitors, possess the ability to reach the iPS cell status. "
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